Method for mass production of plant exosomes

Abstract

The present invention relates to a method for mass production of plant exosomes. The method of the present invention can extract high-purity plant exosomes from a large amount of plant raw materials by using centrifugation and TFF that can simultaneously process a large amount of plant raw materials. The present invention proposes a method that is easy to mass produce by improving the conventional plant exosome extraction method that remains at the laboratory level.

Description

Technical Field

[0001] This application claims priority based on Korean application No. 10-2019-0079490 filed on Jul. 2, 2019, and all contents disclosed in the specification and drawings of the corresponding application are incorporated herein by reference.

[0002] The present disclosure relates to a method for extracting plant raw materials from The present disclosure relates to a method for isolating exosomes from plants having hard pulp or peel. More specifically, the present disclosure relates to a method for large-scale production of high-purity plant exosomes from plant raw materials for useful use in related industries such as pharmaceuticals and commercial products. Background Art

[0003] Exosomes are reported to be nano-sized microvesicles secreted by various cells and contain proteins, lipids, mRNA and miRNA, and act as important mediators of intercellular communication by acting on the endocrine system. This action has attracted attention as a new therapeutic agent that can overcome the problems of conventional stem cell therapeutic agents, such as engraftment rate and immunogenicity.

[0004] According to recent studies, exosomes isolated from edible plants have no toxicity or immunogenicity compared to exosomes secreted from mammalian cells, and are known to have very high in vivo stability and biocompatibility. In addition, exosomes isolated from mammalian cells are reported to contain about 20% cholesterol, but edible plant exosomes do not contain cholesterol. In particular, plant raw materials have the advantage of being available in large quantities compared to animal tissues or cell culture solutions.

[0005] Exosomes are found in a variety of plants depending on the plant of origin, but until now, little was known about the proteins associated with plant exosomes, and there were limitations in characterizing them compared to exosomes from mammalian cells. In most studies, plant exosomes are characterized by their size or shape, and in order to isolate plant exosomes, methods for selecting the appropriate size and isolating only exosomes are needed.

[0006] In the case of the prior art, attempts have been made to separate plant exosomes by density gradient ultracentrifugation using a sucrose gradient, but this method is difficult to use in the commercial production process of exosomes. In addition, there is a method of obtaining substances smaller than the pores of the filtration filter by filtering the separated extracellular vesicles, that is, when separating exosomes from a large amount of plant juice, the filtration efficiency is rapidly reduced because impurities or exosomes are adsorbed and accumulated in the pores of the filter, which is very unfavorable for long-term exosome separation.

[0007] Many documents are cited throughout the specification and their citations are indicated. The disclosures of the cited documents are incorporated herein by reference in their entirety to more clearly illustrate the present disclosure and the level of the art to which the present disclosure belongs.

[0008] Technical issues

[0009] In the present disclosure, a large-scale production method using centrifugation and TFF is suggested as a method for isolating high-purity plant exosomes. Through the present disclosure, the purification and productivity of high-purity plant exosomes are effectively improved.

[0010] Therefore, one object of the present disclosure is to provide a method that can effectively separate high-purity plant exosomes on a large scale, supplementing conventional separation methods.

[0011] Another object of the present disclosure is to provide a method for large-scale isolation of high-purity exosomes with uniform particle size and distribution from plant materials.

[0012] Another object of the present disclosure is to provide a method for isolating plant exosomes which is not limited to plant raw materials including marine flora such as crusts, stems, seeds and sea mustard, laver etc. unlike the prior art methods which are limited to soft flesh or juice.

[0013] Another object of the present disclosure is to provide a method for isolating plant-derived exosomes that is suitable for GMP production of medical and commercial products and facilitates process scale-up.

[0014] Other objects and advantages of the present disclosure will become more apparent from the following detailed description of the invention, the appended claims and the accompanying drawings.

[0015] Technical Solution

[0016] One aspect of the present disclosure is to provide a method for isolating plant exosomes, comprising performing centrifugation; and performing tangential-flow filtration (TFF) as ultrafiltration.

[0017] The term "plant exosome" as used herein refers to an exosome or exosome-like extracellular vesicle having a size of about 50 to 200 nm isolated from a plant.

[0018] Plant-derived exosomes are known to have low toxicity and immunogenicity compared to exosomes secreted from mammalian cells, and have very low cholesterol content, so their stability and biocompatibility are very high. In addition, plant-derived exosomes are known to be effective in wound healing and skin regeneration due to plant-derived antioxidant components or protein components.

[0019] Meanwhile, as various methods for isolating exosomes from living organisms, centrifugation, ultracentrifugation, density gradient centrifugation, chromatography, filtration, ultrafiltration, tangential flow filtration, polymer-based precipitation, total exosome extraction kits, and immunoaffinity separation methods, etc. have been developed. However, in the case of this prior art, the exosome isolation method is limited to a method for isolating exosomes from cells of animal, limited to, mammalian origin or biological solutions (such as milk). Since vacuoles similar to exosomes are found in plant-derived cells different from animal-derived cells, methods for removing them have been introduced, but most are limited to laboratory-level doses.

[0020] In the present disclosure, by specifically combining ultracentrifugation and tangential flow filtration as ultrafiltration among conventionally known exosome separation methods, a method for separating plant-derived exosomes is provided, which can prevent contamination by impurities such as vacuoles and deterioration of purity and obtain high-purity plant exosomes on a large scale. Except for the combination, it is difficult to obtain plant exosomes from which vacuoles are removed by a combination of any other two methods among previously known exosome separation methods, and in particular, it is impossible to uniformly obtain high-purity plant exosomes on a large scale.

[0021] The plant-derived exosomes may have a uniform size distribution of 30 to 500 nm, and preferably, may have a uniform size distribution of 50 to 200 nm.

[0022] In another aspect, in order to replace the conventional density gradient centrifugation method which is limited to the production of plant exosomes in small quantities, a method for large-scale production of plant exosomes using centrifugation and TFF methods is provided.

[0023] Centrifugation used in the present disclosure is a separation method using the density difference between a solid and the liquid surrounding the solid, and refers to a method using centrifugal force to accelerate the sedimentation of the solid. Centrifugation has the advantages of being able to repeat the process continuously, processing a large amount in a short time, and allowing easy operation under aseptic conditions. The centrifugation of the present disclosure can be differential centrifugation, density gradient centrifugation, or a combination thereof, but is not limited thereto.

[0024] The centrifugation method used in the present disclosure can be selected from low-speed centrifugation, high-speed centrifugation, ultracentrifugation and combinations thereof, and preferably, it can include ultracentrifugation. Low-speed centrifugation can reach a speed of 6,000rpm (6,000xg) or lower, and is mainly used for centrifugation of samples that are easy to precipitate, such as cells or cell nuclei, and high-speed centrifugation has a maximum speed of about 20,000 to 25,000rpm (60,000xg), and ultracentrifugation refers to a centrifugation method with a maximum speed of about 40,000 to 80,000rpm (600,000xg).

[0025] Previously, it was known that ultracentrifugation has a low yield and destroys exosomes during the separation process, so it cannot be used for large-scale separation of exosomes and can only be a laboratory-level separation. However, the inventors unexpectedly found that, unlike animal exosomes, plant exosomes can effectively improve productivity by ultracentrifugation without damaging exosomes, and can be applied to the process of mass production of exosomes. In one embodiment, the centrifugation of the present disclosure can be performed by including the following: (a) a process of obtaining a supernatant after centrifugation at a speed of 1,000 to 3,000 x g for 10 to 30 minutes, (b) a process of removing vacuoles by ultracentrifugation at a speed of 10,000 to 50,000 x g for 60 minutes to 2 hours, and (c) a process of obtaining a precipitate containing exosomes by ultracentrifugation at a speed of 100,000 to 150,000 x g for 60 minutes to 2 hours.

[0026] In addition, the method of the present disclosure is characterized in that it includes performing tangential flow filtration (TFF). The tangential flow filtration (TFF) used in the present disclosure is such a filtration method, wherein the solution flows in a direction perpendicular to the filter membrane, filters out small-sized impurities present in the solution, and separates large-sized exosomes. Compared with conventional filtration methods, the adsorption of exosomes to the pores of the filter filter or the clogging of the membrane pores can be minimized, and therefore it is easy to process amplification and GMP process application.

[0027] In TFF, the rapid flow of feed solution serves to reduce concentration polarization (the concentration of product on the pore surface) and to “sweep” the "The effect of the membrane or hollow fiber surface. In addition, the TFF method prevents the increase of contaminants that can clog the pores. This rapid cross-flow reduces the pressure, which can force some feed solutions and dissolved molecules that are smaller than the pores of the membrane or hollow fiber to pass through the filter. The solution that has passed through the pores is called the filtrate or permeate, and the molecules or particles larger than the pores are retained in the feed solution and are effectively concentrated. In a preferred embodiment, the TFF of the present disclosure is an ultrafiltration system, and as a result of performing the TFF method as described above, the centrifuged extract can be effectively concentrated to 1 / 10 to 1 / 100 of the volume. Ultrafiltration is located between microfiltration and reverse osmosis, and is a method of separating specific substances by the size difference between the membrane pores and the solutes. Ultrafiltration membranes can exhibit their separation performance as a molecular weight cutoff (MWCO), which is defined as the minimum molecular weight of the solute that exhibits 90% or more rejection by the membrane.

[0028] The tangential flow filtration used in the present disclosure may be one or more selected from hollow fiber TFF and membrane TFF capable of ultrafiltration, and preferably, it may use a TFF filter having a molecular weight cutoff (MWCO) of 100,000Da to 500,000Da.

[0029] In addition, the method of the present disclosure may further include crushing the plant material before centrifugation and TFF. In this case, the advantage is that the range of plant materials limited to soft pulp can be expanded to hard plants including peels, etc. by mechanical and physical crushing. Therefore, the plant material used in the method of the present disclosure may include one or more selected from the following: pulp, skin, seeds, stems, leaves, roots and flowers.

[0030] Crushing the plant material may include a process of mechanically crushing a mixture in which the plant material and a buffer are mixed in a weight ratio of 1:1 to 1:10, and as the buffer, a buffer containing one or more of phosphate buffered saline (PBS), tris-buffered saline (TBS), 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) buffered saline (HBS), physiological saline solution, distilled water, culture medium, water for injection, etc. can be used, but is not limited thereto.

[0031] The process of crushing the plant material may be performed by mechanically crushing the plant mixture with the blades of a knife rotating at a speed of 10 to 1,000 rpm.

[0032] The extract obtained by the method of the present disclosure may contain a concentration of 10 7 Up to 10 12 The number of particles / 1mL unit volume of plant exosomes.

[0033] The advantage of the method of the present disclosure as described above is that it is suitable for large-scale production of plant exosomes, and can be large-scale processing of 1 mL or more, 10 mL or more, 100 mL or more, 1 L or more, 10 L or more, or 100 L or more of plant materials at a time.

[0034] Effect

[0035] The methods of the present disclosure provide methods for uniformly obtaining high purity exosomes from which vacuoles have been removed. In particular, high purity plant exosomes can be isolated from large amounts of plant raw materials using centrifugation and TFF, which can process large amounts of plant raw materials simultaneously. This improves upon conventional plant exosome isolation methods that remain at the laboratory level, and thereby provides a simple method for large-scale production. BRIEF DESCRIPTION OF THE DRAWINGS

[0036] Figure 1is a schematic diagram of the process of a method for isolating plant-derived exosomes according to one embodiment of the present disclosure.

[0037] Figure 2 This is the morphological analysis (TEM) result of the aloe peel-derived exosomes according to Example 1.

[0038] Figure 3 These are the results of the characterization analysis (NTA) of the exosomes derived from aloe peel according to Example 1.

[0039] Figure 4 This is a TEM photograph comparing the results obtained when the UC process was omitted and only TFF was performed with the results obtained when UC and TFF were combined.

[0040] Figure 5 These are the results of the characterization analysis (DLS) of the exosomes derived from aloe peel according to Example 1.

[0041] Figure 6 These are the results of morphological analysis (TEM) of garlic-derived exosomes according to Example 2.

[0042] Figure 7 These are the results of the characterization analysis (NTA) of garlic-derived exosomes according to Example 2.

[0043] Figure 8 These are the results of morphological analysis (TEM) of the exosomes derived from the sea mustard peel according to Example 3.

[0044] Fig. 9 These are the results of morphological analysis (NTA) of the exosomes derived from the sea mustard peel according to Example 3. DETAILED DESCRIPTION

[0045] Hereinafter, the present disclosure will be described in more detail by way of examples. These examples are only used to illustrate the present disclosure in more detail, and it is obvious to those skilled in the art to which the present disclosure belongs that the scope of the present disclosure is not limited by these examples.

[0046] Example

[0047] 1. Exosomes from aloe vera peel

[0048] (1) Separation method

[0049] -Example 1 (UC+TFF)

[0050] Aloe peel-derived exosomes were separated using UC and TFF. Specifically, aloe peel was placed in a mixer with phosphate buffered saline (PBS) at a ratio of 1:2 (w / w) and then fully ground. Then, the supernatant produced after centrifugation at 1,000xg for 10 minutes was centrifuged at 2000xg for 20 minutes, and the supernatant was collected again. The supernatant was centrifuged at 3,000xg for 30 minutes and then at 10,000xg for 60 minutes. The supernatant after 10,000xg was ultracentrifuged at 100,000xg for 70 minutes at 4°C using UC.

[0051] The precipitate remaining after UC was suspended, and the exosomes were separated from the suspension by a tangential flow filtration (TFF) system. Specifically, other impurity particles smaller than the filter pores were removed from a multi-filter surface with a cutoff value of 100 to 500 kDa, and the solution containing the aloe peel-derived exosomes was concentrated. The exosomes separated in this way were stored frozen at -70°C or lower until used in experiments.

[0052] -Comparative Example 1 (MF+TFF)

[0053] Exosomes derived from aloe peel were separated using microfiltration and TFF. Specifically, aloe peel and phosphate buffered saline (PBS) were set at 1:2 (w / w) and then fully ground in a mixer. Then, in order to obtain high-purity exosomes with uniform particle size distribution, 2% by weight of trehalose was added. After adding trehalose, it was filtered with a 0.22 μm filter to remove impurities such as cell debris, waste and large particles.

[0054] After passing through the filtration process, the exosomes were separated by a tangential flow filtration (TFF) system as ultrafiltration. Specifically, other impurity particles smaller than the filter pores were removed from a multi-filter surface with a cutoff value of 100 to 500 kDa, and the solution containing the aloe peel-derived exosomes was concentrated. The exosomes separated in this way were frozen and stored at a temperature of -70°C or lower until used in experiments.

[0055] (2) Evaluation of exosome characteristics

[0056] For Aloe vera peel derived exosomes, the exosome characteristics were evaluated. To determine the shape of the exosomes, a scanning electron microscope (TEM) was used, and to determine the exact particle size, a dynamic light scattering photometer (DLS) was used. The number of particles of the isolated exosomes per unit volume was determined by nanoparticle tracking analysis (NTA).

[0057] In Example 1, as a result of the experiment, after filtering a solid having a relatively large particle size by microfiltration (pore size 0.1 to 1.0 μm) of the supernatant after centrifugation at 10,000×g, a mixture in which exosomes and vacuoles exist together was obtained, and it was confirmed that exosomes and vacuoles were mixed in the supernatant after centrifugation at 10,000×g. In Example 1, it was confirmed that a large amount of impurities such as proteins and nucleic acids as well as exosomes were mixed in the precipitate collected after performing ultracentrifugation at 100,000×g. In Example 1, as a result of separating exosomes by performing ultrafiltration TFF, 1×10 9～11 It was determined that the isolated aloe peel-derived exosomes had a microstructure of 200 nm or less ( Figure 2 ), and nanoparticle tracking analysis (NTA) determined that 1×10 9～11 The concentration of exosomes / 1mL unit volume ( Figure 3 ).

[0058] On the other hand, it was observed that vacuoles similar to exosomes were mixed in the aloe peel-derived extract separated by the method of Comparative Example 1, and thus it was confirmed that it was difficult to remove vacuoles contained in the plant extract by the MF and TFF methods. In other words, when only TFF was performed without ultracentrifugation (UC) process in the process of separating exosomes from aloe peel, it was clearly confirmed that impurities including vacuoles (red arrows) in addition to exosomes were included, and these impurities caused the efficacy of plant exosomes to decrease (see Figure 4 ).

[0059] 2. Isolation of Garlic-derived Exosomes

[0060] (1) Separation method

[0061] - Example 2 (UC+TFF):

[0062] Garlic-derived exosomes were isolated using UC and TFF. Specifically, garlic with the skin removed was placed in a mixer with phosphate buffered saline (PBS) set at 1:2 (w / w) and then fully ground. Then, the supernatant produced after centrifugation at 1,000xg for 10 minutes was centrifuged at 2,000xg for 20 minutes, and the supernatant was collected again. The supernatant was centrifuged at 3,000xg for 30 minutes and then at 10,000xg for 60 minutes. The supernatant after 10,000xg was ultracentrifuged at 100,000xg for 70 minutes at 4°C using UC. Exosomes were isolated by suspending the precipitate remaining after UC and using a tangential flow filtration (TFF) system as ultrafiltration. Specifically, other impurity particles smaller than the filter pores were removed from the multi-filter surface with a cutoff value of 100 to 500kDa, and the solution containing garlic-derived exosomes was concentrated. The exosomes separated in this way were stored frozen at -70°C or lower until used in experiments.

[0063] -Comparative Example 2 (UC+S-DGUC)

[0064] Garlic-derived exosomes were isolated using UC and sucrose density gradient ultracentrifugation. Specifically, garlic with the skin removed was placed in a mixer with phosphate buffered saline (PBS) set at 1:2 (w / w) and then fully ground. Then, the supernatant produced after centrifugation at 1,000xg for 10 minutes was centrifuged at 2,000xg for 20 minutes, and the supernatant was collected again. The supernatant was centrifuged at 3,000xg for 30 minutes and then at 10,000xg for 60 minutes. The supernatant after 10,000xg was ultracentrifuged at 100,000xg for 70 minutes at 4°C using UC.

[0065] Sucrose density gradient ultracentrifugation was performed separately by suspending the precipitate remaining after UC. Specifically, a sucrose solution with a density gradient was added to a centrifuge tube for ultracentrifugation. These sucrose solutions were made into 90%, 80%, 70%, 60%, 50%, 40% and 30% solutions, respectively, and after sterilization at 124°C for 15 minutes, the tube for ultracentrifugation was carefully placed so that it had a 90% to 30% layer. After the suspension containing the exosomes was carefully added to the top of the sucrose gradient tube for ultracentrifugation prepared as above, ultracentrifugation was performed for 4 hours at 4°C, 200,000xg using a centrifuge for ultracentrifugation. Then, a 1 mL fraction was obtained from the top of the tube, and the density was measured to obtain the fraction corresponding to the exosomes and washed to separate the sucrose.

[0066] (2) Evaluation of exosome characteristics

[0067] As a result of the experiment, it was determined by scanning electron microscopy (TEM) that the garlic-derived exosomes separated in Example 2 had a circular microstructure of 200 nm or less ( Figure 6 ), and as a result of determining the number of particles of isolated exosomes per unit volume by nanoparticle tracking analysis (NTA), 1×10 10～12 The concentration of exosomes / 1mL unit volume ( Figure 7 ).

[0068] The garlic-derived exosomes separated by the method of Comparative Example 2 also had a round microstructure of 200 nm or less, as observed by scanning electron microscopy (TEM). However, in order to separate high-purity exosomes, an additional washing process for separating sucrose was required, and the exosome production per hour was less than 1 / 100 of that of Example 2. Therefore, it was confirmed that the method of Comparative Example 2 was difficult to use for a large-scale production process of exosomes for commercialization.

[0069] 3. Isolation of Exosomes from Sea Mustard

[0070] (1) Separation method

[0071] - Example 3 (UC+TFF):

[0072] UC and TFF were used to separate the exosomes derived from sea mustard. Specifically, the dehydrated sea mustard was placed in a mixer with phosphate buffered saline (PBS) at a ratio of 1:2 (w / w) and then fully ground. Then, the supernatant produced after centrifugation at 1,000xg for 10 minutes was centrifuged at 2,000xg for 20 minutes, and the supernatant was collected again. The supernatant was centrifuged at 3,000xg for 30 minutes and then at 10,000xg for 60 minutes. The supernatant after 10,000xg was ultracentrifuged at 100,000xg for 70 minutes at 4°C using UC.

[0073] Exosomes were isolated by suspending the precipitate remaining after UC and passing it through a tangential flow filtration (TFF) system as ultrafiltration. Specifically, other impurity particles smaller than the filter pores were removed from the surface of a multifilter with a cutoff value of 100 to 500 kDa in the TFF system, and the solution containing the sea mustard-derived exosomes was concentrated. The exosomes isolated in this way were stored frozen at -70°C or lower until used in experiments.

[0074] - Comparative Example 3 (SEC+TFF):

[0075] Size exclusion chromatography is known to be a useful method for separating exosomes without exosome damage or characteristic changes. Therefore, SEC and TFF were used to separate sea mustard-derived exosomes. Specifically, the separation of exosomes by SEC was performed by the method disclosed in Boing et al (Single-step isolation of extracellular vesicles by size-exclusion chromatography, J Extracell Vesicles, 2014: 1-11). 12 mL of Sepharose CL-2B (Sigma Aldrich, St. Louis, MO, USA) was stacked in a 20 mL syringe (BD PlasticpakcTM, San Jose, CA), washed and balanced with PBS. 2 mL of sample was loaded onto the column, and fractions were collected using PBS as the elution buffer.

[0076] Exosomes were separated from the collected fractions by a tangential flow filtration (TFF) system. Specifically, other impurity particles smaller than the filter pores were removed from the surface of a multifilter with a cutoff value of 100 to 500 kDa in the TFF system, and the solution containing the sea mustard-derived exosomes was concentrated. The exosomes separated in this way were stored frozen at -70°C or lower until used in experiments.

[0077] (2) Evaluation of exosome characteristics

[0078] Scanning electron microscopy (TEM) confirmed that the sea mustard-derived exosomes isolated in Example 3 had a circular microstructure of 200 nm or less ( Figure 8 ), and as a result of confirming the number of particles of isolated exosomes per unit volume by nanoparticle tracking analysis (NTA), 1×10 9～11 The concentration of exosomes / 1mL unit volume ( Fig. 9 ).

[0079] On the other hand, it was observed that vacuoles similar to exosomes were mixed in the Seaweed-derived extract separated by the method of Comparative Example 3, and thus it was confirmed that it was difficult to remove vacuoles contained in the plant extract by the SEC and TFF methods.

Claims

1. A method for isolating plant exosomes, comprising: Centrifuging at 1,000 to 3,000 x g for 10 to 30 minutes, and obtaining a supernatant; Centrifuge at 10,000 to 50,000 x g for 60 minutes to 2 hours; Using the supernatant, perform ultracentrifugation at a speed of 100,000 to 150,000 x g for 60 minutes to 2 hours to obtain a precipitate; suspending the precipitate to obtain a suspension; using the suspension to perform tangential flow filtration (TFF); as well as Obtain high-purity plant exosome extracts; The high-purity plant exosome extract contains 10 7 Up to 10 12 particles / 1 mL of the plant exosomes of the extract but not containing vacuoles, The tangential flow filtration uses a TFF filter with a molecular weight cut-off (MWCO) of 100,000 Da to 500,000 Da. 2 . The method for separating plant exosomes according to claim 1 , wherein the tangential flow filtration is one or more selected from the group consisting of hollow fiber TFF and membrane TFF.

3. The method for isolating plant exosomes according to claim 1 or 2, wherein the method further comprises crushing the plant material before the centrifugation.

4. The method for isolating plant exosomes according to claim 3, wherein the plant raw material comprises one or more selected from the group consisting of pulp, skin, seeds, stems, leaves, roots and flowers. 5 . The method for isolating plant exosomes according to claim 1 , wherein the diameter of the plant exosomes is 50 to 200 nm.

6. The method for isolating plant exosomes according to claim 1 or 2, wherein the high-purity plant exosome extract contains a concentration of 1×10 9-11 The plant exosomes contain 100 particles / 1 mL of the extract.

7. The method for isolating plant exosomes according to claim 1 or 2, wherein the method is used for large-scale production of plant-derived exosomes.

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